Hepatitis B virus (HBV) infection is a serious global health problem affecting approximately 2 billion people worldwide. About 400 million people infected by HBV are chronic carriers. Each year, about 1 million people die of various HBV diseases such as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) (World Health Organization, 2000). The disease is relatively rare in Western countries, acquired mainly in adulthood. However, the disease is virtually endemic in Asian and African countries, where most chronic HBV infections are acquired perinatally or during childhood.
The hepatitis B virus (HBV) is a small DNA virus of about 3200 bases with extensive sequence variability. Currently eight genotypes (A to H) are recognized.
The virus can cause debilitating disease conditions. Acute infection may lead to liver failure, although it is rare. Hepatitis B infection develops into a chronic form in over 90% of those infected at birth or as infants. Chronic infection eventually lead to cirrhosis and carcinoma of the liver in 25-40% of the patients. HBV is estimated to cause 30% of cirrhosis and 50% of HCC globally (Perz et al., 2006). This enormous burden highlights the need for the development of effective antiviral therapy, which is useful in reducing the risk of HCC (Liaw et al, 2004). It is therefore important to monitor a patient's response to such therapy.
HBV is mainly spread vertically with the virus passed on from infected mothers to infants at birth. Infants can also be infected through close contact with infected parents and siblings. HBV is also spread horizontally by sexual contacts or close contact with infected blood.
The virus replicates via an RNA intermediate and utilizes reverse transcription that lacks proofreading capability in its replication strategy. The HBV genome is partially double stranded and encodes envelope, precore/core, reverse transcriptase/polymerase and X genes in four overlapping open reading frames.
The envelope protein antigen is referred to as HBsAg (hepatitis B surface antigen) and it makes up the outer surface coat of the virus. The core protein antigen is referred to as HBcAg (hepatitis B core antigen) and it forms the core of the virus that encapsulates the HBV DNA. The precore protein antigen is referred to as HBeAg (hepatitis B e antigen) with unknown function. The protein X antigen is referred to as HBxAg (hepatitis B x antigen) and its exact function is also unknown.
The polymerase gene overlaps the envelope gene. Thus, mutations affecting the catalytic domain of the polymerase gene can also affect the protein sequence of the envelope protein and vice versa.
Current treatments for chronic hepatitis B infection include interferon, and nucleoside/nucleotide analogues. Unfortunately, treatment with interferon has many side effects and only a small proportion of patients respond to therapy.
Nucleoside or nucleotide analogues are chemically engineered nucleotides developed to act as substitute building blocks to inhibit viral DNA synthesis during viral replication. Currently approved (in the US) nucleoside and nucleotide analogues for treatment of chronic hepatitis B are: lamivudine (3TC or LMV), telbivudine (L-dT), entecavir (ETV), adefovir (ADV) and tenofovir.
While these compounds have been effective at inhibiting HBV DNA synthesis, they require long-term treatment and there is the potential for resistant mutant HBV strains to emerge during prolonged treatment. In patients on long-term treatment with LMV, common resistance conferring mutations are rtM204I/V+/−rtL180M (Allen et al., 1998) as well as other mutations. In some patients, resistant viral strains carrying mutations in the B, C and D domains of the HBV DNA polymerase gene emerge upon prolonged therapy. These patients then have a higher risk of developing HCC, compared to LMV-treated patients without resistant virus.
Additionally, during the process of hepatitis B e-antigen (HBeAg) seroconversion, precore and core promoter mutants emerge due to selection under immune pressure. These precore mutants have been implicated as the cause of more severe HBV infections. Core promoter mutants, whilst causing a decrease in precore mRNA transcription and HBeAg production, enhance viral replication, are also related to the development of HCC.
Kazim et al. (2006) report that core promoter and YMDD motif mutations are associated with viral breakthrough in patients on long-term LMV therapy. Also, variants of the gene coding for the surface antigen has been found in HBV vaccinated children, in post liver transplantation patients receiving anti-HBV immunoglobulin therapy, in patients with occult infection (Weber, 2005) and in patients treated with LMV (Torresi et al., 2002).
The protection of HBV vaccination is based on the induction of antibodies against major antigenic epitopes of the HBV surface-antigen (HBsAg). For patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease, prophylaxis against recurrent HBV infection is given by administration of hepatitis B immunoglobulins derived from vaccinated subjects. Again, the emergence of immune escape HBV mutants results in viral persistence in spite of adequate antibody titers.
The development of effective antiviral therapy therefore requires a method for monitoring the emergence of resistant strains of HBV and to develop assays to detect and identify these resistant viruses so that the clinician can make a timely switch to a different treatment regimen when resistance arises.
Many studies have addressed the prevalence of different genotypes throughout the world (Lindh, M, Anderson A S, Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus—large-scale analysis using a new genotyping method. J Infect Dis 1997; 175:1285-1293; Chan H L Y, Tsui S K W, Tse C-H, Ng E Y T, Au T C C, Yuen L, Bartholomeusz A, Leung K-S, Lee K-H, Locarnini S, Sung J J Y. Epidemiological and virological characteristics of 2 subgroups of hepatitis B virus genotype C. J Infect Dis 2005; 191:2022-2032; and Yuen M-F, Sablon E, Yuan H-J, Wong D K-H, Hui C-K, Wong B C-Y, Chan A O-O, Lai C L. Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma. Hepatology 2003; 37:562-567) and the predisposition of different genotypes to disease complications such as development of cirrhosis, HCC (Kuang S Y, Jackson P E, Wang J-B, Lu P-X, Munoz A, Qian G-S, Kensler T W, Groopman J D. Specific mutations of hepatitis B virus in plasma predict liver cancer development. Proc Natl Acad Sci, USA 2004; 101:3575-3580; and Yuen M F, Sablon E, Wong D K H, Yuan H-J, Wong B C-Y, Chan A O O, Lai C L. Role of hepatitis B virus genotypes in chronic hepatitis B exacerbation. Clin Infect Dis 2003; 37:593-597) and response to treatment (Kao J H, Chen P J, Lai M Y, Chen D S. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000; 118:554-559; and Buti M, Cotrina M, Valdes A, Jardi R, Rodriguez-Frias F, Esteban R. Is hepatitis B virus subtype testing useful in predicting virological response and resistance to lamivudine? J Hepatol 2002; 36:445-446)
To aid in the management of hepatitis B patients, various test strips based on reverse dot blot (RDB) allele-specific oligonucleotide (ASO) hybridization technique were developed to identify either genotype (INNO-LiPA HBV genotyping. Innogenetics NV, Belgium) or mutations (INNO-LiPA HBV precore. Innogenetics NV, Belgium; and INNO-LiPA DR. Innogenetics NV, Belgium). Since RDB-ASO principle requires the same hybridization and washing conditions for all variants under analysis, all normal and mutant probes would need similar melting temperature (Tm) (Saiki R K, Walsh P S, Levenson C H, Erlich H A. Genetic analysis of amplified DNA with an immobilised sequence-specific oligonucleotide probe. Proc Natl Acad Sci, USA. 1989; 86:6230-6234). This poses limitations on testing >20 mutations/genotypes on a single RDB strip. Currently three separate test strips are needed to assess eight genotypes and 12 mutations (INNO-LiPA HBV genotyping. Innogenetics NV, Belgium; INNO-LiPA HBV precore. Innogenetics NV, Belgium; and INNO-LiPA DR. Innogenetics NV, Belgium). Other groups have devised oligonucleotide microarray/chip (H, Cho M, Hco J, Kim H, Jun H, Shin W, Cho B, Park H, Kim C. Oligonucleotide chip for detection of lamivudine-resistant Hepatitis B Virus. J Clin Microbiol 2004; 42:4181-4188; Mao H, Wang H, Zhang D, Mao H, Zhao J, Shi J, Cui Z. Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray. Clin Biochem 2006; 39:67-73; Song Y, Dai E, Wang J, Liu H, Zhai J, Chen C, Du Z, Guo Z, Yang R. Genotyping of hepatitis B virus (HBV) by oligonucleotides microarray. Mol Cell Probes. Epub 2006 January; and Li Z G, Chen L Y, Huang J, Qiao P, Qiu J M, Wang S Q. Quantification of the relative levels of wild-type and lamivudine-resistant mutant virus in serum of HBV-infected patients using microarray. J Viral Hepatitis 2005, 12:168-175), but the same RDB-ASO principle was employed. The maximum number of mutations detectable was 20.